The objective of this study is to elucidate the biochemical and electrophysiological mechanisms which lead to an irreversible loss of cardiac cell excitability under oxidative stress. Single cardiac cells isolated from rabbit ventricles and will be subjected to increasing levels of oxidative stress produced by exposure to either tertiary-butylhydroperoxide or Fenton reaction generated hydroxyl radicals. The types of free radicals generated will be identified and quantitated by electron spin resonance. In order to isolate the metabolic components of oxidative stress induced myocardial injury, glucose metabolism of the cell ,,i glycolysis, oxidative phosphorylation and hexose monophosphate shunt will be studied at different states of oxidative stress. Experiments will be performed to examine how alterations in the intracellular levels of ATP, NAD(P)H, glutathione and calcium, produced by oxidative stress lead to impaired membrane excitability. Whole cell and cell attached configurations of the giga seal patch clamp technique will be used co assess the alterations in the ion transport pathways of the myocardial cell membrane, specifically the alterations in the Na channel activity and the causes of membrane depolarization, including the involvement of the Na pump. The molecular mechanisms of the oxidant induced alterations in ion permeability will be examined in order [o understand whether oxidative stress affects channel and/or transport proteins directly, by oxidation, proteolysis and lipid peroxidation or indirectly as a consequence of alterations in the biochemical processes that regulate channel function. In the case of the Na channel, immunological techniques in conjunction with electrophysiological studies will be used to identify the specific mechanism(s) of oxidation induced changes. These studies should permit an identification of the sequence of events that precede and lead to the loss of membrane excitability and to uncover the interrelationships between these events. A comprehensive understanding of the mechanisms that lead to oxidative damage should be of long term significance in explaining oxidative stress induced cardiac tissue damage and dysfunctions that may be produced on exposure to xenobiotics and during clinical ischemia-reperfusion episodes.